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Primary research Close relationship of tissue plasminogen activator–plasminogen activator inhibitor-1 complex with multiple organ dysfunction syndrome investigated by means of the artificial pancreas Masami Hoshino*, Yoshikura Haraguchi † , Hiroyuki Hirasawa ‡ , Motohiro Sakai*, Hiroshi Saegusa*, Kazushiro Hayashi*, Naoki Horita* and Hiroyuki Ohsawa* *Department of Intensive and Critical Care Medicine, Tokyo Police Hospital, Chiyoda-ku, Tokyo, Japan † National Hospital Tokyo Disaster Medical Center, Tachikawa-shi, Tokyo, Japan ‡ Department of Emergency and Critical Care Medicine, Chiba University School of Medicine, Chuo-ku, Chiba-shi, Chiba, Japan Correspondence: Masami Hoshino, Department of Intensive and Critical Care Medicine, Tokyo Police Hospital, Fujimi 2-10-41, Chiyoda-ku, Tokyo 102, Japan. Tel: +81 3 3263 1371; fax: +81 3 3239 7856; e-mail: noel2000@aioros.ocn.ne.jp AP = artificial pancreas; AT-III = antithrombin III; BG = blood glucose level; DIC = disseminated intravascular coagulation; ECA = endothelial cell activation; ECI = endothelial cell injury; GT = glucose tolerance; MODS = multiple organ dysfunction syndrome; mMOF = modified multiple organ failure; NIDDM = noninsulin-dependent diabetes mellitus; PAI-1 = plasminogen activator inhibitor-1; PLT = platelet count; PT = prothrombin time; SRH = stress related hormone; TAT = thrombin–antithrombin III complex; TM = thrombomodulin; tPA = tissue plasminogen activator; T 3 = triiodothyronine; T 4 = thyroxine. Available online http://ccforum.com/content/5/2/088 Abstract Background: Glucose tolerance (GT) has not been taken into consideration in investigations concerning relationships between coagulopathy and multiple organ dysfunction syndrome (MODS), and endothelial cell activation/endothelial cell injury (ECA/ECI) in septic patients, although coagulopathy is known to be influenced by blood glucose level. We investigated those relationships under strict blood glucose control and evaluation of GT with the glucose clamp method by means of the artificial pancreas in nine septic patients with glucose intolerance. The relationships between GT and blood stress related hormone levels (SRH) were also investigated. Methods: The amount of metabolized glucose (M value), as the parameter of GT, was measured by the euglycemic hyperinsulinemic glucose clamp method, in which the blood glucose level was clamped at 80 mg/dl under a continuous insulin infusion rate of 1.12 mU/kg per min, using the artificial pancreas, STG-22. Multiple organ failure (MOF) score was calculated using the MOF criteria of Japanese Association for Critical Care Medicine. Regarding coagulopathy, the following parameters were used: disseminated intravascular coagulation (DIC) score (calculated from the DIC criteria of the Ministry of Health and Welfare of Japan) and the parameters used for calculating DIC score, protein-C, protein-S, plasminogen, antithrombin III (AT-III), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator–PAI-1 (tPA-PAI-1) complex. Thrombomodulin (TM) was measured as the indicator of ECI. Results: There were no significant correlations between M value and SRH, parameters indicating coagulopathy and the MOF score. The MOF score and blood TM levels were positively correlated with DIC score, thrombin–AT-III complex and tPA-PAI-1 complex, and negatively correlated with blood platelet count. Conclusions: GT was not significantly related to SRH, coagulopathy and MODS under strict blood glucose control. Hypercoagulability was closely related to MODS and ECI. Among the parameters indicating coagulopathy, tPA-PAI-1 complex, which is considered to originate from ECA, seemed to be Received: 1 December 1998 Revisions requested: 17 April 2000 Revisions received: 1 June 2000 Accepted: 18 November 2000 Published: 26 February 2001 Critical Care 2001, 5:88–99 This article may contain supplementary data which can only be found online at http://ccforum.com/content/5/2/088 © 2001 Hoshino et al, licensee BioMed Central Ltd (Print ISSN 1364-8535; Online ISSN 1466-609X) Available online http://ccforum.com/content/5/2/088 commentary review reports meeting abstracts primary research supplement Introduction Hypercoagulability and decreased fibrinolysis, including increased PAI-1 level, are often found in the clinical field and are considered to be the risk factors of cardiovascu- lar diseases and glucose intolerance, especially in patients with noninsulin-dependent diabetes mellitus (NIDDM) [1–6]. Most of the acutely ill severe patients also have coagulopathy, and they often have glucose intolerance. The relationships between coagulopathy and organ dysfunction/glucose intolerance in the acute ill phase have not, however, been clearly analyzed. Although there are reports investigating the relationship between coagulopathy and organ dysfunction [7–13], and the relationship between coagulopathy and endothe- lial cell activation/injury [14–17] in septic patients, there is no report investigating the relationship between coag- ulopathy and GT in septic patients as far as we know. Moreover, parameters related to coagulopathy are known to be influenced directly by the metabolic factors. For example, glucose, insulin, and fat influence the pro- duction of PAI-1, which is the important parameter related to coagulopathy [18–23]. In aforementioned reports, however, metabolic factors, especially blood glucose level (BG) that is usually unstable in the septic state, are not taken into consideration. We have been using the bedside type artificial pancreas (AP) in septic patients with glucose intolerance since 1985 to control BG, to perform effective nutritional support, and to evaluate metabolic disorders including glucose and fat. By strictly stabilizing BG using AP, analy- ses of the factors including PAI-1 that are influenced by BG are considered to be correctly performed. The purpose of this study is, first, to analyze the relation- ships between coagulopathy, including abnormal blood PAI-1-related parameters, and glucose tolerance, MODS, and endothelial cell injury. Second was to investigate which parameters related to coagulopathy were most closely related to glucose tolerance, MODS, and endothelial cell injury, in septic patients with glucose intolerance in whom BG was strictly controlled and the glucose tolerance was evaluated with the glucose clamp method by means of AP. We consider that better understanding of the aforemen- tioned relationships and confirming the useful parameters will be helpful for the early diagnosis of the severity of sepsis and for the treatment of the septic patients. Materials and methods The investigated patients were nine septic intensive care unit patients with glucose intolerance in whom BG was strictly controlled by means of AP. We selected the patients with strict blood glucose control by AP in order to exclude the direct influence of BG to the parameters related with coagulopathy, including PAI-1-related para- meters, as already mentioned. The patients were all in septic condition, which was defined as the condition with systemic inflammatory response syndrome caused by the infection [24]. To analyze the septic patients with sepsis- induced (or related) glucose intolerance, the diabetes patients and those who had liver or pancreatic diseases as primary diseases were excluded. Six patients had acute respiratory distress syndrome (four caused by panperitoni- tis, two after intracranial hemorrhage), two had gangrene of a lower extremity, and one had a burn (Table 1). One patient with panperitonitis died. Regarding administered drugs that might influence glucose tolerance on the day when the GT measurements were performed (total number of measurements, 18 times (days); 2 times (days) for each patient; see later), dopamine was used for 5 patients (6 days out of 10 mea- sured days), predonisolone for 1 patient (2 days out of 2 measured days), and dobutamine for 1 patient (2 days out of 2 measured days). The amount of dopamine used was less than 5 µg/kg per min (mean, 2.5 ± 1.6 µg/kg per min [n = 6]; all were used for increasing renal blood flow), that of predonisolone was 40 mg/day, and that of dobutamine was 13 and 4 µg/kg per min. Analyzed items were as follows. Regarding MODS, the multiple organ failure (MOF) score was calculated using the MOF criteria of the Japanese Association for Critical Care Medicine [25] (Table 2). The maximum of the MOF score is 14. The modified MOF score (mMOF score), in which points of disseminated intravascular coagulation (DIC) (coagulopathy) were excluded, was also calculated when the correlation between coagulopathy and MODS was investigated. The parameter of glucose metabolism, the M value (the amount of metabolized glucose), was measured by the euglycemic hyperinsulinemic glucose clamp method, in which the BG level was clamped (or maintained) at 80 mg/dl under a continuous insulin infusion rate of a sensitive parameter of MODS and ECI, and might be a predictive marker of MODS. The treatment for reducing hypercoagulability and ECA/ECI were thought to be justified as one of the therapies for acutely ill septic patients. Keywords: artificial pancreas, coagulopathy, diabetes mellitus, multiple organ dysfunction syndrome, tissue plasminogen activator-plasminogen activator inhibitor-1 complex Critical Care Vol 5 No 2 Hoshino et al 1.12 mU/kg per min (40 mU/m 2 per min), using AP. The M value is the amount of glucose infusion required to clamp BG, and is the indicator of peripheral glucose tolerance (normal range, 6–8 mg/kg per min) [26,27]. The daily mean blood glucose level was calculated from the BG measured (sampled) every 1 h. Table 1 Primary diseases of the nine septic patients with glucose intolerance Patient Age (years) Sex Diseases Prognosis 1 54 F ARDS (after panperitonitis due to perforation of the ileum) Alive 2 67 M ARDS (after panperitonitis due to NOMI) Alive 3 74 M ARDS (after panperitonitis due to perforation of the duodenum) Died 4 49 M ARDS (after panperitonitis due to necrotic cholecystitis) Alive 5 47 M ARDS (after the operation of acute subdural hematoma) Alive 6 59 M ARDS (after the operation of subarachnoideal hemorrhage) Alive 7 66 M Gangrene of lower extremity Alive 8 74 M Gangrene of lower extremity Alive 9 21 M Burn Alive ARDS, Acute respiratory distress syndrome; F, female; M, male; NOMI, nonocclusive mesenteric ischemia. Table 2 Multiple organ failure score calculated using the criteria proposed by the Japanese Association for Critical Care Medicine [25] Impaired organ Criteria Points Impaired organ Criteria Points Kidney Urine output <600 ml/day; or 50 mg/dl < BUN; 1 Digestive tract Hematemesis, melena; or ulcer; 1 or 3–5 mg/dl creatinine or blood transfusion greater than 2 U/day 5 mg/dl < creatinine; or 0 ml/h < CH 2 O; 2 bleeding from digestive tract with hypotension, 2 or 3.0% < FENa or perforation, necrosis Lung PaO 2 <60 mmHg (room air); or 250 mmHg 1 Brain 10–100 JCS, or 8–12 GCS 1 ≤ PaO 2 /FiO 2 <350 mmHg; or 300–400 mmHg A-aDO 2 (FiO 2 = 1.0); or 20–30% Qs/Qt; 100<JCS, or 8<GCS, or convulsion with 2 or with respirator for more than 5 days unconsciousness, or no auditory brain stem response, or brain death PaO 2 /FiO 2 <250 mmHg; or 400 mmHg 2 < A-aDO 2 (FiO 2 = 1.0); or 30%<Qs/Qt DIC 20 µg/ml ≤ FDP; or platelet ≤ 80,000/µl; 1 or fibrinogen ≤100 mg/dl; or exacerbation Liver 3.0–5.0 mg/dl bilirubin; or 100 IU/l < s-GPT; 1 of FDP, platelet, fibrinogen within 2 days or 0.4–0.7 AKBR 1 (more than three times greater than or one-third of normal value), or with heparin 5.0 mg/dl < bilirubin; or AKBR < 0.4 2 (≥50 U/kg per day) or probable DIC Cardiovascular 10 mmHg < CVP, or major arrhythmia, 1 Definite DIC 2 or Forrester classification: peripheral vascular resistance < 1000 dyne s/cm 5 ; or with inotropic agents for more than 2 h Forrester classification: with shock, or life 2 threatening arrythmia, or acute myocardial infarction, or cardiac arrest, or major arrhythmia with shock Judgement of probable disseminated intravascular coagulation (DIC)* and definite DIC* from Criteria of DIC proposed by the Ministry of Health and Welfare of Japan [28]. A-aDO 2 , alveolar–arterial oxygen difference; AKBR, arterial ketone body ratio; BUN, blood urea nitrogen; CVP, central venous pressure; FDP, fibrin and fibrinogen degradation products; FENa, fractional excretion of sodium; GCS, Glasgow coma scale; JPS, Japan coma scale. The blood concentration of stress hormones (cate- cholamines [adrenaline, noradrenaline, dopamine], growth hormone, glucagon, cortisol), adrenocorticotrophic hormone, and thyroid-related hormones (thyroid stimulat- ing hormone, triiodothyronine [T 3 ], free T 3 , thyroxine [T 4 ], free T 4 ) were measured because they might influence the GT. Dopamine, dobutamine, and predonisolone were administered when the measurements of the M value were performed in some patients as already mentioned. Regarding coagulopathy, the following parameters were used: DIC score, platelet count (PLT), fibrin and fibrinogen degradation products, fibrinogen, prothrombin time (PT) ratio (PT of the patient divided by control PT), D-dimer, α 2 plasmin inhibitor–plasmin complex, thrombin–antithrombin III complex (TAT), protein C antigen and activity, protein S antigen and activity, plasminogen, antithrombin III (AT-III), PAI-1 antigen and activity, and tissue plasminogen activator (tPA)–PAI-1 complex. The DIC score was calculated from the DIC criteria of the Ministry of Health and Welfare of Japan [28] (Table 3). As the indicator of the endothelial cell injury, the blood con- centration of thrombomodulin (TM) was measured. Fibrino- gen was measured by the thrombin time method, PT by Quick’s method, and fibrin and fibrinogen degradation prod- ucts by the latex agglutination method. TAT, tPA–PAI-1 complex, protein S antigen, protein S activity and TM were measured by enzyme immunoassay, D-dimer and PAI-1 Available online http://ccforum.com/content/5/2/088 commentary review reports meeting abstracts primary research supplement Table 3 Criteria of disseminated intravascular coagulation (DIC) (The Ministry of Health and Welfare of Japan [28]) Criteria Points 1. Underlined disease (+) 1 (–) 0 2. Symptom (1) Bleeding tendency (+) 1 (–) 0 (2) Symptom caused by organ dysfunction (+) 1 (–) 0 3. Laboratory data (1) Serum FDP (µg/ml) 40 ≤ 3 20 ≤ < 40 2 10 ≤ < 20 1 10 > 0 (2) Platelet (× 10 3 /µl) 50 ≥ 3 80 ≥ > 50 2 120 ≥ > 80 1 120 0 (3) Plasma fibrinogen (mg/dl) 100 ≥ 2 150 ≥ > 100 1 150 < 0 (4) Prothrombin time/control 1.67 ≤ 2 1.25 ≤ < 1.67 1 1.25 > 0 4. Supplemental data (1) Detection of soluble fibrin monomer (2) Increase of D-dimer (3) Increase of thrombin–antithrombin complex (4) Increase of plasmin-a 2 –plasmin inhibitor complex (5) Exacerbation of FDP, platelet, fibrinogen within several days (6) Improvement of data with anticoagulant therapy Judgment* 1. Definite DIC (1) Patients who do not have leukemia, pernicious anemia, liver cirrhosis, More than 7 or 6 points with or who are not under cancer chemotherapy more than two of supplemental data (2) Patients who have leukemia, pernicious anemia, or who are More than 4 or 3 points with under cancer chemotherapy points for bleeding tendency and platelet more than two of supplemental data are not included (3) Patients who have liver cirrhosis More than 10 or 9 points with more than two of supplemental data 2. Probable DIC (1) Patients who do not have leukemia, pernicious anemia, liver cirrhosis, 6 points or who are not under cancer chemotherapy (2) Patients who have leukemia, pernicious anemia, or who are under 3 points cancer chemotherapy points for bleeding tendency and platelet are not included (3) Patients who have liver cirrhosis 9 points *Exclusion: this DIC criteria cannot be applied for neonates, pregnant woman, and patients with fulminant hepatitis. FDP, fibrin and fibrinogen degradation products. antigen by enzyme-linked immunosorbent assay, and α 2 plasmin inhibitor–plasmin complex and protein C antigen by the Latex photometric immunoassay. AT-III, PAI-1 activity and plasminogen were measured by the synthetic substrate method, and protein C activity by the activated partial throm- boplastin time method (SRL Inc Co, Tokyo, Japan). Data sampling/measurement (blood sampling, MOF/DIC scoring, and glucose clamp method) was performed twice for each patient. The first data sampling/measurement was carried out within 3 days after the admission, and the second data sampling/measurement was performed 1 week after the first data sampling/measurement. Blood sampling was carried out at 08:00 h on the day when the glucose clamp method (the measurement of the M value) was performed. We began the glucose clamp method at 09:00 h, when intravenous drip infusion containing glucose for nutritional support was stopped. The daily mean blood glucose level was calculated using the BG during 24 h before the start of the glucose clamp method. The following points were investigated in turn using the aforementioned data. First, confirmation of the capability of the AP for strict blood glucose control (by calculating the daily mean BG) and for the evaluation of the GT (M value). Whether the blood concentration of the stress-related hor- mones (listed earlier), which are considered to be influenced by sepsis and by the administration of drugs, was related to the GT (M value) was also investigated. Third, whether there were any relationships between the glucose tolerance (M value) and coagulopathy, MODS (MOF score). The relation- ships among coagulopathy, MODS (MOF/mMOF score), and endothelial cell injury (TM) were then investigated. Finally, confirmation of the parameters related to coagulopa- thy that were most closely correlated with MODS (MOF/mMOF score) and endothelial cell injury (TM). The AP used was STG-22, manufactured by NIKKISOH Corporation (Tokyo, Japan) (Fig. 1). The AP controls BG by administering insulin or glucose automatically according to the absolute BG and the change of BG, which is measured by continuous blood sampling. The statistical data are shown as mean ± standard devia- tion. Strengths of the relationships between the data are indicated by correlation coefficient r, and the correlations between the data are shown by a regression line. The unpaired Student t test was used for the comparison of mean values. P < 0.05 was considered significant. Results Blood glucose control and measurements of the glucose tolerance by means of AP The mean of the daily mean blood glucose levels and M values obtained from the first and second measurements were 183 ± 32 mg/dl (n = 8), 4.4 ± 1.4 mg/kg per min (n = 7), and 147 ± 26 mg/dl (n = 9), 4.7 ± 1.6 mg/kg per min (n = 8), respectively (Table 4). The daily mean blood glucose level could not be calculated in one patient at the first measurement because a blood sampling disorder of AP occurred and a sufficient number of BG data could not be obtained. M values could also not be measured in two patients at the first measurement and in one patient at the second measurement because the glucose intolerance was severe and the BG level did not decrease to the clamp level (80 mg/dl). No significant relationships between the GT and blood stress related hormone levels There were no significant correlations between the M value and blood stress hormone levels (adrenaline, nora- drenaline, dopamine, growth hormone, glucagon, cortisol), adrenocorticotrophic hormone, and thyroid-related hor- mones (thyroid stimulating hormone, T 3 , free T 3 , T 4 , free T 4 ) (Table 5). We also investigated whether drug (cate- cholamines [dopamine, dobutamine], glucocorticoids [pre- Critical Care Vol 5 No 2 Hoshino et al Figure 1 Bedside-type artificial pancreas STG-22. donisolone]) administration significantly influenced the glucose tolerance. There was, however, no significant dif- ference between the mean of the M values of the patients who were administered those drugs (4.9 ± 1.3 mg/kg per min; n = 10) and that of those who were not administered those drugs (4.0 ± 1.7 mg/kg per min; n = 5). No significant correlations between the GT and MODS, coagulopathy There were no significant correlations between the M value and MODS, and parameters related to coagulation and fibrinolysis (Table 6). Significant correlation between coagulopathy and MODS The MOF score was strongly correlated with the DIC score (r = 0.75, P < 0.002), TAT (r = 0.72, P < 0.002), tPA–PAI- 1 complex (r = 0.69, P < 0.002) and PLT (r = –0.68, P < .002) among parameters related with coagulation and fibrinolysis (Table 7). Because three of the aforementioned parameters (not the tPA–PAI-1 complex) are used for cal- culating the MOF score, however, correlations between the mMOF score, in which the points of coagulopathy of the MOF score are excluded, and parameters related to coagu- lation and fibrinolysis were also analyzed. The mMOF score was still strongly correlated with TAT (r = 0.69, P < 0.002), DIC score (r = 0.66, P < 0.002), PLT (r = –0.65, P < 0.003) and tPA–PAI-1 complex (r = 0.62, P < 0.005) (Table 8; Fig. 2). Significant correlations between endothelial cell injury and MODS, coagulopathy TM was correlated with MOF score (r = 0.92, P < 0.002) (Fig. 3), DIC score (r = 0.80, P < 0.002), tPA–PAI-1 complex (r = 0.85, P < 0.002), TAT (r = 0.85, P < 0.002) and PLT (r = –0.58, P < 0.03) (Table 9; Fig. 4). In one patient, the measurement of TM was performed only once. Relationships between tPA–PAI-1 complex and other parameters related to coagulation and fibrinolysis The tPA–PAI-1 complex was positively correlated with DIC score (r = 0.74, P < 0.002), TAT (r = 0.85, P < 0.002), and PAI-1 antigen (Table 10). Available online http://ccforum.com/content/5/2/088 commentary review reports meeting abstracts primary research supplement Table 4 Blood glucose control and measurements of glucose tolerance by means of artificial pancreas Daily mean blood glucose levels (mg/dl) Mean M values (mg/kg per min) First measurement 183 ± 32 (n = 8) 4.4 ± 1.4 (n = 7) Second measurement 147 ± 26 (n = 9) 4.7 ± 1.6 (n = 8) Total 164 ± 34 (n = 17) 4.6 ± 1.5 (n = 15) The first measurement was performed within 3 days after admission, and the second measurement was performed 1 week after the first measurement. Table 5 No significant correlations between glucose tolerance and blood stress related hormone levels: correlation coefficient ( r ) between the M value and hormones Normal range Mean r P Adrenaline (ng/ml) ≤0.17 0.11 ± 0.10 (n = 18) –0.17 < 0.55 (n = 15) Noradrenaline (ng/ml) 0.15–5.7 0.79 ± 0.74 (n = 18) 0.15 < 0.60 (n = 15) Dopamine (ng/ml) ≤0.03 19 ± 41 (n = 18) 0.20 < 0.48 (n = 15) Growth hormone (ng/ml) 0.28–8.70 4.7 ± 5.2 (n = 18) –0.45 < 0.09 (n = 15) Glucagon (pg/ml) 23–197 234 ± 156 (n = 18) 0.39 < 0.17 (n = 14) Cortisol (µg/dl) 5.6–21.3 33 ± 42 (n = 18) –0.10 < 0.73 (n = 15) ACTH (pg/ml) ≤60 28 ± 19 (n = 18) –0.05 < 0.87 (n = 15) TSH (µU/ml) 0.5–4.8 1.1 ± 1.6 (n = 17) 0.49 < 0.08 (n = 14) T 3 (ng/dl) 80–180 82 ± 30 (n = 17) –0.05 < 0.87 (n = 14) Free T 3 (pg/ml) 2.5–4.5 2.2 ± 0.7 (n = 17) –0.10 < 0.74 (n = 14) T 4 (µg/dl) 5.0–13.7 9.4 ± 4.2 (n = 17) 0.09 < 0.76 (n = 14) Free T 4 (ng/dl) 0.8–1.9 1.4 ± 0.6 (n = 17) 0.11 < 0.71 (n = 14) ACTH, Adrenocorticotrophic hormone; TSH, thyroid stimulating hormone; T 3 , triiodothyronine; T 4 = thyroxine. Critical Care Vol 5 No 2 Hoshino et al Table 6 No significant correlations between glucose tolerance and multiple organ dysfunction syndrome, coagulopathy: correlation coefficient ( r ) between the M value and the multiple organ failure (MOF) score/parameters related with coagulopathy Normal range Mean rP MOF score 4.6 ± 1.5 (n = 15) 0.20 < 0.48 (n = 15) DIC score 4.0 ± 1.8 (n = 18) 0.32 < 0.25 (n = 15) PLT (/µl) 150,000–280,000 188,000±123,000 (n = 18) 0.39 < 0.15 (n = 15) FDP (µg/ml) < 10 14 ± 15 (n = 18) 0.49 < 0.06 (n = 15) PT ratio < 1.25 1.3 ± 0.10 (n = 16) –0.02 < 0.95 (n = 13) Fibrinogen (mg/dl) 150–350 500 ± 154 (n = 18) –0.23 < 0.42 (n = 15) TAT (ng/ml) ≤3.0 13.2 ± 13.0 (n = 18) 0.34 < 0.22 (n = 15) PIC (µg/ml) ≤0.8 1.2 ± 0.7 (n = 18) 0.27 < 0.34 (n = 15) D-Dimer (ng/ml) ≤150 750 ± 630 (n = 18) 0.43 < 0.11 (n = 15) Plasminogen (%) 75–125 80 ± 23 (n = 18) 0.25 < 0.37 (n = 15) AT-III (%) 70–120 94 ± 28 (n = 13) 0.25 < 0.47 (n = 11) Protein C activity (%) 55–140 75 ± 43 (n = 18) 0.44 < 0.10 (n = 15) Protein C antigen (%) 70–150 92 ± 51 (n = 18) 0.47 < 0.08 (n = 15) Protein S activity (%) 60–150 72 ± 18 (n = 18) 0.42 < 0.12 (n = 15) Protein S antigen (%) 65–135 83 ± 24 (n = 18) 0.42 < 0.12 (n = 15) PAI-1 activity (U/ml) 12–15 5.3 ± 3.4 (n = 13) 0.55 < 0.10 (n = 10) PAI-1 antigen (ng/ml) ≤50 120 ± 86 (n = 18) 0.22 < 0.44 (n = 15) tPA–PAI-1 complex (ng/ml) ≤11 26 ± 18 (n = 18) 0.27 < 0.34 (n = 15) AT-III, antithrombin-III; DIC, disseminated intravascular coagulation; FDP, fibrin and fibrinogen degradation products; PAI-1, plasminogen activator inhibitor-1; PIC, α 2 plasmin inhibitor–plasmin complex; PLT, platelet count; PT, prothrombin time; TAT, thrombin–antithrombin complex; tPA, tissue plasminogen activator. Table 7 Correlation coefficients ( r ) between multiple organ failure score and parameters related to coagulation and fibrinolysis rPn DIC score 0.75 < 0.002 18 TAT 0.72 < 0.002 18 tPA–PAI-1 complex 0.69 < 0.002 18 PLT –0.68 < 0.002 18 Protein S activity –0.48 < 0.04 18 Plasminogen –0.43 < 008 18 Protein C activity –0.41 < 0.09 18 D-Dimer 0.38 < 0.12 18 FDP 0.37 < 0.13 18 Protein C antigen –0.31 < 0.22 18 Protein S antigen –0.25 < 0.32 18 PAI-1 activity –0.25 < 0.42 13 PT ratio –0.23 < 0.40 16 AT-III 0.22 < 0.48 13 PIC 0.2 < 0.43 18 PAI-1 antigen 0.19 < 0.46 18 Fibrinogen 0.000,09 < 1.0 18 AT, Antithrombin; DIC, disseminated intravascular coagulation; FDP, fibrin and fibrinogen degradation products; PAI-1, plasminogen activator inhibitor-1; PIC, α 2 plasmin inhibitor–plasmin complex; PLT, platelet count; PT, prothrombin time; TAT, thrombin–antithrombin complex; tPA, tissue plasminogen activator. Table 8 Correlation coefficients ( r ) between modified multiple organ failure (mMOF) score* and parameters related to coagulation and fibrinolysis r P n DIC score 0.66 < 0.002 18 TAT 0.69 < 0.002 18 PLT –0.65 < 0.003 18 tPA–PAI-1 complex 0.62 < 0.005 18 Protein S activity –0.44 < 0.07 18 Protein C activity –0.43 < 0.08 18 Plasminogen –0.42 < 0.08 18 Protein C antigen –0.32 < 0.20 18 D-Dimer 0.3 < 0.23 18 AT-III 0.24 < 0.46 13 Protein S antigen –0.22 < 0.38 18 PAI-1 activity –0.21 < 0.50 13 PT ratio –0.18 < 0.51 16 PAI-1 antigen 0.18 < 0.48 18 PIC 0.16 < 0.54 18 Fibrinogen 0.000,03 < 1.0 18 * mMOF score = MOF score – points of coagulopathy of the MOF score. AT, Antithrombin; DIC, disseminated intravascular coagulation; PAI-1, plasminogen activator inhibitor-1; PIC, α 2 plasmin inhibitor–plasmin complex; PLT, platelet count; PT, prothrombin time; TAT, thrombin–antithrombin complex; tPA, tissue plasminogen activator. Discussion Acutely ill patients often have coagulopathy and meta- bolic disorders, including glucose intolerance and abnor- mal serum fat levels, as well as organ dysfunctions. Those abnormalities seem to be mutually related, but studies concerning relationships among coagulopathy, metabolic disorders, and organ dysfunctions have rarely been reported. One of the reasons for this lack of litera- ture seems to be that metabolic disorders, especially glucose intolerance, are unstable and could not be easily evaluated in acute phase. In this study, we have investi- gated those relationships under strict blood glucose control and the strict evaluation of the GT with the glucose clamp method by means of AP in septic patients with glucose intolerance. Although the glucose tolerances of the patients were impaired, blood glucose control by means of AP was good, considering results of the mean of the M values and the daily mean BG (Table 4). We could not measure the M value three times because the GT was so severe that BG did not decrease to the clamp level (80 mg/dl). This problem was considered to indicate the necessity of the improvement for measuring the M value in patients with severe GT (eg increasing the amount of insulin infu- Available online http://ccforum.com/content/5/2/088 commentary review reports meeting abstracts primary research supplement Figure 2 Correlations between mMOF score and parameters related to coagulation and fibrinolysis. The mMOF score (mMOF score = MOF score – the points of coagulopathy of MOF score) was positively correlated with (a) the DIC score, (b) TAT and (d) tPA–PAI-1 complex, and (c) negatively correlated with PLT. Figure 3 Correlation between TM and MOF score. The MOF score was positively correlated with blood TM level. sion, stopping intravenous drip infusion earlier than 09:00 h, etc). There are many factors that influence BG or the GT. Stress hormones and thyroid-related hormones are well known to be included in those factors, and they are also used as the drugs. In the present study in which the AP strictly con- trolled BG, however, those hormones did not significantly influence the glucose tolerance. This is determined from the results that there were no significant correlations between the M value and the blood concentration of these hormones (Table 5), and that there were no significant differences in the M values between the patients who were administered these hormones and those who were not. We consider that sepsis induced by some other factors other than these hormones impaired the glucose tolerance. The relationship between GT including fat metabolism and hypercoagulability, indicated by the increased levels of PAI-1 or tPA–PAI-1 complex, has been well investigated in the patients with NIDDM [5,29,30], with hypertension [31–34], with coronary artery disease [35], and in the normal human subjects or the general population [36,37]. In these studies, PAI-1 or tPA–PAI complex was closely related with, and thought to be caused by, hyperinsuline- mia, hyperglycemia, insulin resistance, hypertriglyc- eridemia, hypercholesterolemia, and increased level of high density lipoprotein cholesterol. In vitro studies using endothelial cells, hepatoma cells, or vascular smooth muscle cells showed that PAI-1 was produced by glucose, insulin, free fatty acid, cholesterol, very low density lipoprotein, glucocorticoids, and hyperosmolarity [18–23]. In our study performed under strict blood glucose control by means of AP, however, the glucose intolerance was not a significant factor influencing MODS and coagulopathy, considering from the results that there were no significant correlations between the M value and the MOF score, parameters related with coagulation and fibrinolysis (Table 6). In addition, under this strict blood glucose control, BG, blood insulin and fat levels did not significantly influence the coagulopathy, because there were no significant correlations between parameters related with coagulation and fibrinolysis and daily mean BG, blood insulin concentration, and serum fat (triglyc- eride, total cholesterol, free fatty acid) levels (data not shown). These results are considered to indicate that the Critical Care Vol 5 No 2 Hoshino et al Figure 4 Correlations between TM and parameters related with coagulation and fibrinolysis. Blood TM levels were positively correlated with (a) the DIC score, (b) tPA–PAI-1 complex and (c) TAT, and (d) negatively correlated with PLT. influence of the GT and the factors related with the glucose tolerance (eg BG, blood insulin and fat levels) to coagulopathy could be excluded by the strict blood glucose control using AP. Relationships between coagulopathy and chronic organ dysfunctions have been well investigated. The hypercoag- ulable state or decreased fibrinolytic activity in NIDDM patients, shown by increased levels of PAI-1, fibrinogen, factor VII, von Willebrand factor, and tPA, are considered to be risk factors of cardiovascular diseases [1–6,29, 38,39]. Increased PAI-1 level is especially thought to be a causative factor of atherosclerosis [5,6,38]. In patients other than those with NIDDM, including those with insulin- dependent diabetes mellitus [40], history of myocardial infarction [41], and hypertension [31–33], hypercoagula- ble states with increased PAI-1 level are also considered to be one of the risk factors of coronary atherosclerosis or hypertension. Increased PAI-1 level seems to be the cause of, and not only the result of, cardiovascular dis- eases or atherosclerosis, because it was shown in an animal study that increased expression of PAI-1 in the arterial wall preceded atherosclerosis [6]. Relationships between hypercoagulable state and sepsis or septic MODS have been investigated in recent years [7–13]. The hypercoagulable state, shown by increased levels of PAI-1 [7,8,10,13], TAT [7–9], and prothrombin fragment 1 + 2 [11], and by decreased levels of AT-III [7,9,11,12], factor VII [7,11], and protein C [12], were reported in these studies to be closely related to septic MODS. As mentioned in the Introduction, however, meta- bolic factors including glucose and fat that are considered to influence those parameters related with coagulopathy are not taken into consideration in those investigations. In our study, performed with strict blood glucose control by AP, the MOF score (mMOF score) was positively corre- lated with the DIC score, TAT, and tPA–PAI-1 complex, and was negatively correlated with PLT (Tables 7 and 8; Fig. 2). The tPA–PAI-1 complex, which is reported to posi- tively correlate with tPA [42–45], is considered to be a parameter of hypercoagulability and decreased fibrinoly- sis, and to be closely related with thrombotic diseases [42,43]. The tPA–PAI-1 complex was in fact also posi- tively related with TAT (Table 10) in this study, which is the parameter of hypercoagulability. On the contrary, there were no significant correlations between the MOF score (mMOF score) and parameters related with fibrinolysis (α 2 plasmin inhibitor–plasmin complex, fibrin and fibrinogen degradation products, D-dimer). Judging from the afore- mentioned results in the present study, hypercoagulability and decreased fibrinolysis, indicated by the increase of Available online http://ccforum.com/content/5/2/088 commentary review reports meeting abstracts primary research supplement Table 9 Correlation coefficients ( r ) between thrombomodulin (TM) and multiple organ failure (MOF) score, parameters related to coagulation and fibrinolysis* rPn MOF score 0.92 < 0.002 17 DIC score 0.80 < 0.002 17 tPA–PAI-1 complex 0.85 < 0.002 17 TAT 0.85 < 0.002 17 PLT –0.58 < 0.01 17 FDP 0.50 < 0.04 17 D-Dimer 0.48 < 0.05 17 PAI-1 antigen 0.40 < 0.11 17 Protein C activity –0.38 < 0.13 17 Protein S activity –0.36 < 0.16 17 PIC 0.33 < 0.20 17 Fibrinogen –0.31 < 0.23 17 Plasminogen –0.31 < 0.23 17 Protein C antigen –0.3 < 0.25 17 AT-III 0.27 < 0.41 12 PT ratio –0.25 < 0.38 15 Protein S antigen –0.15 < 0.57 17 PAI-1 activity –0.04 < 0.90 12 * Mean of TM, 7.3 ± 4.2 FU/ml (n = 17); normal range, ≤4.5. AT-III, antithrombin-III; DIC, disseminated intravascular coagulation; FDP, fibrin and fibrinogen degradation products; PAI-1, plasminogen activator inhibitor-1; PIC, α 2 plasmin inhibitor–plasmin complex; PLT, platelet count; PT, prothrombin time; TAT, thrombin–antithrombin complex; tPA, tissue plasminogen activator. Table 10 Correlation coefficients ( r ) between the tissue plasminogen activator–plasminogen activator inhibitor-1 (tPA–PAI-1) complex and other parameters related to coagulation and fibrinolysis r P n DIC score 0.74 < 0.002 18 TAT 0.85 < 0.002 18 PAI-1 antigen 0.60 < 0.007 18 PLT –0.49 < 0.04 18 Protein C activity –0.39 < 0.11 18 PT ratio –0.37 < 0.16 18 D-Dimer 0.36 < 0.14 18 FDP 0.36 < 0.14 18 Protein S antigen –0.34 < 0.17 18 Protein C antigen –0.33 < 0.18 18 Plasminogen –0.31 < 0.22 18 PIC 0.30 <0.23 18 Fibrinogen –0.10 < 0.70 18 AT-III 0.035 < 0.90 13 PAI-1 activity –0.014 < 0.98 13 AT-III, antithrombin-III; DIC, disseminated intravascular coagulation; FDP, fibrin and fibrinogen degradation products; PIC, α 2 plasmin inhibitor–plasmin complex; PLT, platelet count; PT, prothrombin time; TAT, thrombin–antithrombin complex. [...]... related to the origin of the parameters PAI-1 is synthesized not only by the endothelium, but also by the liver, vascular smooth muscle cells, and platelets [46] AT-III is synthesized by the liver and endothelium, and protein C by the liver On the contrary, the tPA–PAI-1 complex is considered to be synthesized mainly by the activated endothelium, because the tPA–PAI-1 complex is the indicator of tPA,... intimate relationship between organ dysfunction and endothelial cell activation Endothelial cell injury was closely related with MODS and coagulopathy characterized by hypercoagulability with decreased fibrinolysis, judging from the results that TM was closely correlated with MOF score, DIC score, tPA–PAI-1 complex, TAT, and PLT (Table 9; Figs 3 and 4) These results are consistent with the other reports... injury, and might be one of the predictive and risk factors of MODS Finally, the treatment for reducing hypercoagulability and endothelial cell activation/endothelial cell injury was thought to be justified as one of the therapies for acutely ill septic patients Further investigation will, however, be necessary for clarifying these conclusions because the number of the patients we investigated was limited... tPA–PAI-1 complex (Table 9), which was considered to be one of the parameters of endothelial cell activation as mentioned earlier Conclusion We investigated acutely ill septic patients with glucose intolerance in which BG was strictly controlled and the glucose tolerance was measured by the glucose clamp method by means of AP, and obtained the following conclusions The GT did not significantly relate with. .. coagulopathy and MODS under strict blood glucose control Coagulopathy characterized by hypercoagulability with decreased fibrinolysis was closely related with MODS and endothelial cell injury Among the parameters related with coagulation and fibrinolysis, the tPA–PAI-1 complex, considered to originate from activated endothelium, seemed to be a sensitive parameter of MODS and endothelial cell injury, and... already mentioned, and tPA is synthesized by the endothelium activated with thrombin, cytokines (eg tumor necrosis factor, interleukin-2), endotoxin, endothelin, catecholamine, histamine, and activated protein C [17,47,48] A positive correlation between MOF score (mMOF score) and tPA–PAI-1 complex therefore suggests not only a close relationship between organ dysfunction and hypercoagulability, but... S: Genetic variation at the plasminogen activator inhibitor-1 locus is associated with altered levels of plasminogen activator inhibitor-1 activity Arterioscler Thromb 1991, 11:183–190 Nordt TK, Klassen KJ, Schneider DJ, Sobel BE: Augumentation of synthesis of plasminogen activator inhibitor type-1 in arterial endothelial cells by glucose and its implications for local fibrinolysis Arterioscler Thromb... 1996, 39:1425–1431 Schneider DJ, Sobel BE: Synergistic augumentation of expression of plasminogen activator inhibitor type-1 induced by insulin, very-low-density lipoproteins, and fatty acids Coronary Artery Dis 1996, 7:813–817 Chen YQ, Su M, Walia RR, Hao Q, Covington JW, Vaughan DE: Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promotor by glucose in vascular smooth muscle cells... reporting that the hypercoagulable state preceded MODS, in which significant changes of the parameters related with hypercoagulability were found at the onset of sepsis [7] We also found that changes of the tPA–PAI-1 complex preceded those of organ dysfunctions in some cases (data not shown) These findings suggest that the hypercoagulable state disturbs microcirculation and leads to MODS [8,10] There were... Hoshino et al the tPA–PAI-1 complex and TAT, were considered to be closely related with MODS in acutely ill septic patients The tPA–PAI-1 complex, which is not used for calculating the MOF score and the DIC score, also seemed to be a useful and sensitive marker of MODS Moreover, a hypercoagulable state indicated by the elevated tPA–PAI-1 level may be one of the risk factors and predictive markers of MODS . Primary research Close relationship of tissue plasminogen activator plasminogen activator inhibitor-1 complex with multiple organ dysfunction syndrome investigated by means of the artificial. is synthesized by the liver and endothelium, and protein C by the liver. On the contrary, the tPA–PAI-1 complex is considered to be synthesized mainly by the activated endothelium, because the. related to the limited number of the patients in our study, or related to the origin of the parameters. PAI-1 is synthesized not only by the endothelium, but also by the liver, vascular smooth muscle